ICMS was performed using an IZ2 stimulator controlled by an RZ5D base processor (Tucker-Davis Technologies, FL, US). Each pulse consisted of a 200 µs cathodic leading phase followed by a 200 µs anodic phase, with balanced charges between the cathodic and anodic phases. The current was fixed to ±20 µA (4 nC/phase), selected to be lower than the safety limit (k = log(Q) + log(Q/A) ≈ 0.36 < 1.7, where Q and Q/A represent charge per phase [µC/phase] and charge density per phase [µC cm−2/phase], and value k below 1.7 can be considered as a safe configuration [44 (link)]). Six different train frequencies (10, 25, 50, 100, 250, and 500 Hz) were tested. The order of ICMS trials was repeated two times in ascending-descending frequency order (10–500, 500–10, 10–500, 500–10 Hz).
For comparison, a single blue LED was positioned in front of the animal’s eye contralateral to the imaging window as a more natural visual stimulus (n = 5 out of 8 mice in the mesoscopic-scale widefield imaging, n = 5 out of 9 mice in the two-photon imaging). The LED was controlled by the stimulator, applying a square wave at 10 Hz (50 ms on, 50 ms off, 50% duty cycle) with an amplitude of 2.5 V. All stimulation trials were repeated 4 times.
For comparison, a single blue LED was positioned in front of the animal’s eye contralateral to the imaging window as a more natural visual stimulus (n = 5 out of 8 mice in the mesoscopic-scale widefield imaging, n = 5 out of 9 mice in the two-photon imaging). The LED was controlled by the stimulator, applying a square wave at 10 Hz (50 ms on, 50 ms off, 50% duty cycle) with an amplitude of 2.5 V. All stimulation trials were repeated 4 times.